Fluorescent dye compounds, conjugates and uses thereof

ABSTRACT

The present teachings generally relate to fluorescent dyes, linkable forms of fluorescent dyes, energy transfer dyes, reagents labeled with fluorescent dyes and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/165,575, filed Jun. 21, 2011, now allowed; which is a continuation ofU.S. application Ser. No. 11/838,793, filed Aug. 14, 2007, now U.S. Pat.No. 7,985,602; which is a continuation of U.S. application Ser. No.11/229,012, filed Sep. 16, 2005, now U.S. Pat. No. 7,256,292; whichclaims a priority benefit under 35 U.S.C. §119(e) from U.S. PatentApplication No. 60/611,119, filed Sep. 16, 2004; all of which areincorporated herein by reference.

The present teachings generally relate to fluorescent dyes, linkableforms of fluorescent dyes, energy transfer dyes, and reagents labeledwith fluorescent dyes, and uses thereof.

The non-radioactive detection of biological analytes utilizingfluorescent labels is an important technology in modern molecularbiology. By eliminating the need for radioactive labels, safety isenhanced and the environmental impact and costs associated with reagentdisposal is greatly reduced. Examples of methods utilizing suchnon-radioactive fluorescent detection include 4-color automated DNAsequencing, oligonucleotide hybridization methods, and detection ofpolymerase-chain-reaction products, immunoassays, and the like.

In many applications it is advantageous to employ multiple spectrallydistinguishable fluorescent labels in order to achieve independentdetection of a plurality of spatially overlapping analytes, e.g.,single-tube multiplex DNA probe assays and 4-color automated DNAsequencing methods. In the case of multiplex DNA probe assays, byemploying spectrally distinguishable fluorescent labels, the number ofreaction tubes may be reduced thereby simplifying experimental protocolsand facilitating the production of application-specific reagent kits. Inthe case of 4-color automated DNA sequencing, multicolor fluorescentlabeling allows for the analysis of multiple bases in a single lanethereby increasing throughput over single-color methods and reducinguncertainties associated with inter-lane electrophoretic mobilityvariations.

Currently available multiplex dye sets suitable in 4-color automated DNAsequencing applications require blue or blue-green laser light toadequately excite fluorescence emissions from all of the dyes making upthe set, e.g., argon-ion lasers. Use of blue or blue-green lasers incommercial automated DNA sequencing systems are often disadvantageousbecause of the high cost and limited lifetime of such lasers.

Thus, there exists a need for fluorescent dye compounds that satisfy theabove constraints and are excitable by light having a wavelength aboveabout 600 nm.

It has now been found that red fluorescence emitting dyes based on thestructure (1) are very chemically and photoactively stable and areexcitable by light of longer wavelengths.

In some embodiments, the present teachings provide novel fluorescentdyes comprising a structure selected from,

wherein

R₁-R₃ and R₆-R₁₆ can each independently be —H, halogen, fluorine,chlorine, bromine, aryl, substituted aryl, heteroaryl, —CO₂H, —CO₂R,—SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂, —CH₂NHR,—NO₂, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substitutedC₁-C₆ alkoxy, C₁-C₆ alkoxyaryl, substituted C₁-C₆ alkoxyaryl, phenyl,substituted phenyl, biphenyl, substituted biphenyl, benzyl, substitutedbenzyl, benzoyl, substituted benzoyl, bond or linking group, wherein Rcan be C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substitutedC₁-C₆ alkoxy, C₁-C₆ alkoxyaryl, substituted C₁-C₆ alkoxyaryl, phenyl,substituted phenyl, biphenyl, substituted biphenyl, benzyl, substitutedbenzyl, benzoyl, substituted benzoyl, bond or linking group; and

R₄ and R₅ taken separately can be C₁-C₆ alkyl, C₁-C₆ substituted alkyl,R₄ and R₅ taken together can be C₃-C₇ cycloalkyl, C₄-C₇ unsaturatedcycloalkyl, C₃-C₇ substituted cycloalkyl or C₄-C₇ substitutedunsaturated cycloalkyl;

with the proviso that if the compound comprises the structure (I), thenat least one of R₁-R₃ or R₈ is not —H. Optionally, at least one of R₁-R₃and R₆-R₁₆ can be —SO₃H.

In some embodiments, the present teachings provide for energy transferdye compounds comprising a donor dye covalently attached to an acceptordye, wherein the donor dye is capable of absorbing light at a firstwavelength and emitting excitation energy in response, and the acceptordye is capable of absorbing the excitation energy emitted by the donordye and fluorescing at a second wavelength in response. In someembodiments, the donor dye can be covalently attached to the acceptordye by a bond, a non-nucleotidic linker or a nucleotidic linker (i.e.—apolynucleotide, ribonucleic acid, and the like). In some embodiments,the linker can serve to facilitate efficient transfer of energy betweenthe donor dye and the acceptor dye. In some embodiments, at least one ofthe donor and acceptor dyes is a dye of the present teachings.

In some embodiments, the present teachings provide for labelednucleosides and/or nucleotides comprising the structureNUC-L-D

wherein NUC comprises a nucleoside, a nucleotide, a modified nucleosideor a modified nucleotide, L comprises a bond or a linker and D comprisesa dye compound of the present teachings. In some embodiments, NUC and Dcan be covalently linked by a linking moiety, L, wherein L can beattached to D at one of R₁-R₃ and R₆-R₁₆. In some embodiments, if NUCcomprises a purine base, the linking moiety can be attached to the8-position of the purine, if NUC comprises a 7-deazapurine base, thelinking moiety can be attached to the 7-position of the 7-deazapurine,and if NUC comprises a pyrimidine base, the linking moiety can beattached to the 5-position of the pyrimidine.

In some embodiments, the present teachings provide for oligonucleotideanalysis methods comprising the steps of forming a set of labeledoligonucleotide fragments labeled with a dye of the structure set forthabove, subjecting the labeled oligonucleotide fragments to asize-dependent separation process, e.g., electrophoresis, and detectingthe labeled oligonucleotide fragments subsequent to the separationprocess.

These and other features and advantages of the present teachings willbecome better understood with reference to the following description,figures, and appended claims.

FIG. 1 shows a generalized synthetic pathway for the synthesis of dyecompounds of the present teachings comprising the structure (I).

FIG. 2 shows a generalized synthetic pathway for the synthesis of dyecompounds of the present teachings comprising the structure (II).

FIG. 3 shows a generalized synthetic pathway for the synthesis of dyecompounds of the present teachings comprising the structure (III).

FIG. 4 shows a generalized synthetic pathway for the synthesis of dyecompounds of the present teachings comprising the structure (IV).

FIG. 5 shows a possible synthetic pathway for the synthesis of dyecompounds of the present teachings comprising the structure (I).

FIG. 6 shows an exemplary synthetic pathway for the synthesis of atertiary alcohol (e.g. —2-(4′-hydroxynathalen-2-yl)-2-propanol)intermediate useful for the preparation of compounds of the presentteachings.

FIG. 7 shows a possible synthetic scheme for the preparation ofcompounds of the present teachings comprising the structure (II).

FIG. 8 shows possible synthetic schemes for the preparation of compoundsof the present teachings comprising the structure (II).

FIG. 9 shows absorption spectra of DDAO and several representativecompounds of the present teachings.

FIG. 10 shows emission spectra of DDAO and several representativecompounds of the present teachings.

Reference will now be made in detail to alternative embodiments of thepresent teachings, examples of which are illustrated in the accompanyingdrawings. While the present teachings will be described in conjunctionwith the alternative embodiments, it will be understood that they arenot intended to limit the present teachings to those embodiments. On thecontrary, the present teachings are intended to cover all alternatives,modifications, and equivalents, which may be included within theinvention as defined by the appended claims.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“comprising,” as well as other forms, such as “comprises” and“comprise,” will be considered inclusive, in that the term “comprising”leaves open the possibility of including additional elements.

It will be understood that the chemical structures that are used todefine compounds of the present teachings are each representations ofone of the possible resonance structures that each given structure canbe represented by. Further, it will be understood that by definition,resonance structures are merely a graphical representation used by thoseof skill in the art to represent electron delocalization, and that thepresent teachings are not limited in any way by showing one particularresonance structure for a given structure.

Generally, the present teachings comprise fluorescent dye compoundsuseful as fluorescent labels, as components of energy transfer dyes, inconjugates of nucleosides, nucleotides and polynucleotides, in methodsutilizing such dyes and reagents in the area of analyticalbiotechnology. The compounds of the present teachings may findparticular application in the area of fluorescent nucleic acid analysis,e.g., automated DNA sequencing and fragment analysis, detection of probehybridization in hybridization arrays, detection of nucleic acidamplification products, and the like.

In some embodiments, the present teachings provide novel fluorescentdyes comprising a structure selected from,

wherein

R₁-R₃ and R₆-R₁₆ can each independently be —H, halogen, fluorine,chlorine, bromine, aryl, substituted aryl, heteroaryl, —CO₂H, —CO₂R,—SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂, —CH₂NHR,—NO₂, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substitutedC₁-C₆ alkoxy, C₁-C₆ alkoxyaryl, substituted C₁-C₆ alkoxyaryl, phenyl,substituted phenyl, biphenyl, substituted biphenyl, benzyl, substitutedbenzyl, benzoyl, substituted benzoyl, bond or linking group, wherein Rcan be C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substitutedC₁-C₆ alkoxy, C₁-C₆ alkoxyaryl, substituted C₁-C₆ alkoxyaryl, phenyl,substituted phenyl, biphenyl, substituted biphenyl, benzyl, substitutedbenzyl, benzoyl, substituted benzoyl, bond or linking group; and

R₄ and R₅ taken separately can be C₁-C₆ alkyl, C₁-C₆ substituted alkyl,R₄ and R₅ taken together can be C₃-C₇ cycloalkyl, C₄-C₇ unsaturatedcycloalkyl, C₃-C₇ substituted cycloalkyl or C₄-C₇ substitutedunsaturated cycloalkyl;

with the proviso that if the compound comprises the structure (I), thenat least one of R₁-R₃ or R₈ is not —H. Optionally, at least one of R₁-R₃and R₆-R₁₆ can be —SO₃H. It will be understood that any of the compoundsdescribed herein can include the phenol oxygen deprotonated form as wellas all possible resonance structures.

In some embodiments, dye compounds of the present teachings can comprisethe structure:

In some embodiments, dye compounds of the present teachings can comprisethe structure:

In some embodiments, dye compounds of the present teachings can comprisethe structure:

In some embodiments, dye compounds of the present teachings can comprisethe structure:

In some embodiments, R₆ and R₇ can each independently be halogen,fluorine, chlorine or bromine. In some embodiments, R₆ and R₇ can befluorine. In some embodiments, R₆ and R₇ can be chlorine. In someembodiments, R₆ and R₇ can be bromine.

In some embodiments, R₆ can be —H and R₇ can be —H, —CO₂H, —CO₂R, —SO₃H,—SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂, and —CH₂NHRwhere R is defined as above. In some embodiments, X₁ can be —H and X₂can be —H, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂NH₂, and —CH₂NHR whereR is define as above.

In some embodiments, dye compounds of the present teachings comprise thestructure (I), wherein R₁-R₃ and R₈ can each independently be —H, —CO₂H,—CO₂R, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂,—CH₂NHR or —NO₂, wherein R is defined as above, with the proviso that ifthe compound comprises the structure (I), then at least one of R₁-R₃ orR₈ is not —H. In some embodiments, R₁-R₃ and R₈ can each independentlybe —SO₃H, —SO₃R, —CH₂NH₂, —CH₂NHR or —NO₂, wherein R is defined asabove. In some embodiments, R₁-R₃ and R₈ can each independently be —SO₃Hor —SO₃R, wherein R is defined as above. In some embodiments, R₁-R₃ andR₈ can each independently be —CH₂NH₂ or —CH₂NHR, wherein R is defined asabove.

In some embodiments, dye compounds of the present teachings comprise thestructure (I), wherein R₆ and R₇ can each independently be halogen,fluorine, chlorine or bromine and R₁-R₃ and R₈ can each independently be—H, —CO₂H, —CO₂R, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R,—CH₂NH₂, —CH₂NHR, —NO₂, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆alkoxy, substituted C₁-C₆ alkoxy, C₁-C₆ alkoxyaryl, substituted C₁-C₆alkoxyaryl, phenyl, substituted phenyl, biphenyl, substituted biphenyl,benzyl, substituted benzyl, benzoyl, substituted benzoyl, bond orlinking group, wherein R is defined as above, with the proviso that ifthe compound comprises the structure (I), then at least one of R₁-R₃ orR₈ is not —H. In some embodiments, R₆ and R₇ can each independently behalogen, fluorine, chlorine or bromine and R₁-R₃ and R₈ can eachindependently be —SO₃H, —SO₃R, —CH₂NH₂, —CH₂NHR or —NO₂, wherein R isdefined as above. In some embodiments, R₆ and R₇ are fluorine and R₁-R₃and R₈ can each independently be —SO₃H, —SO₃R, —CH₂NH₂, —CH₂NHR or —NO₂,wherein R is defined as above. In some embodiments, R₆ and R₇ arechlorine and R₁-R₃ and R₈ can each independently be —SO₃H, —SO₃R,—CH₂NH₂, —CH₂NHR or —NO₂, wherein R is defined as above. In someembodiments, R₆ and R₇ are bromine and R₁-R₃ and R₈ can eachindependently be —SO₃H, —SO₃R, —CH₂NH₂, —CH₂NHR or —NO₂, wherein R isdefined as above.

In some embodiments, dye compounds of the present teachings can comprisethe structure (I), wherein R₆ can be —H and R₇ can be —H, —SO₃H, —SO₃R,—CH₂CO₂H, —CH₂CO₂R, —CH₂NH₂, or —CH₂NHR, wherein R is defined as above,and R₁-R₃ and R₈ can each independently be —SO₃H, —SO₃R, —CH₂NH₂,—CH₂NHR, or —NO₂, wherein R is defined as above. In some embodiments, R₆can be —H and R₇ can be —H, —SO₃H or —SO₃R, and R₁-R₃ and R₈ can eachindependently be —CH₂NH₂ or —CH₂NHR, wherein R is defined as above.

In some embodiments, a dye compound of the present teachings cancomprise the structure

In some embodiments, a dye compound of the present teachings cancomprise the structure

In some embodiments, a dye compound of the present teachings cancomprise the structure

In some embodiments, R can be substituted benzoyl. In some embodiments,R can be linking group. In some embodiments, R can be trifluoroacetyl.In some embodiments, R can comprise the structure

In some embodiments, R can comprise the structure

X can be succinimide.

In some embodiments, R can be

In some embodiments, R₄ and R₅ can be methyl. In some embodiments, atleast one of R₂ and R₃ can be —NO₂ and R₄ and R₅ can be methyl.

In some embodiments, dye compounds of the present teachings can comprisethe structure (II), wherein R₆ and R₇ can each independently be halogen,fluorine, chlorine or bromine, R can be —CO₂H, —CO₂R, —SO₃H, —SO₃R,—CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂, —CH₂NHR or —NO₂. Insome embodiments, dye compounds of the present teachings can comprisethe structure (II), wherein R₆ and R₇ can each independently be halogen,fluorine, chlorine or bromine and any of R₃, R₈ and R₉-R₁₂ can be —CO₂H,—CO₂R, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂,—CH₂NHR, —NO₂, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,substituted C₁-C₆ alkoxy, C₁-C₆ alkoxyaryl, substituted C₁-C₆alkoxyaryl, phenyl, substituted phenyl, biphenyl, substituted biphenyl,benzyl, substituted benzyl, benzoyl, substituted benzoyl, bond orlinking group, wherein R is defined as above.

In some embodiments, dye compounds of the present teachings can comprisethe structure (II), wherein R₆ and R₇ can each independently be halogen,fluorine, chlorine or bromine, R₉ can be —CO₂H, —CO₂R, —SO₃H, —SO₃R,—CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂, —CH₂NHR or —NO₂, andR₁₀-R₁₂ can be —H, wherein R is defined as above. In some embodiments,dye compounds of the present teachings can comprise the structure (II),wherein R₆ and R₇ can each independently be halogen, fluorine, chlorineor bromine, R₁₀ can be —CO₂H, —CO₂R, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R,—CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂, —CH₂NHR or —NO₂, and R₉ and R₁₁-R₁₂ can be—H, wherein R is defined as above. In some embodiments, dye compounds ofthe present teachings can comprise the structure (II), wherein R₆ and R₇can each independently be halogen, fluorine, chlorine or bromine, R₁₁can be —CO₂H, —CO₂R, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H,—CH₂SO₃R, —CH₂NH₂, —CH₂NHR or —NO₂, and R₉-R₁₀ and R₁₂ can be —H,wherein R is defined as above. In some embodiments, dye compounds of thepresent teachings can comprise the structure (II), wherein R₆ and R₇ caneach independently be halogen, fluorine, chlorine or bromine, R₁₂ can be—CO₂H, —CO₂R, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R,—CH₂NH₂, —CH₂NHR or —NO₂, and R₉-R₁₁ can be —H, wherein R is defined asabove.

In some embodiments, dye compounds of the present teachings can comprisethe structure (II), wherein R₆ can be —H and R₇ can be —H, —SO₃H, —SO₃R,—CH₂CO₂H, —CH₂CO₂R, —CH₂NH₂, or —CH₂NHR, wherein R is defined as above,R₉ can be —CO₂H, —CO₂R, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H,—CH₂SO₃R, —CH₂NH₂, —CH₂NHR or —NO₂ and R₁₀-R₁₂ can be —H, wherein R isdefined as above. In some embodiments, dye compounds of the presentteachings can comprise the structure (II), wherein R₆ can be —H and R₇can be —H, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂NH₂, or —CH₂NHR,wherein R is defined as above, R₁₀ can be —CO₂H, —CO₂R, —SO₃H, —SO₃R,—CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂, —CH₂NHR or —NO₂, and R₉and R₁₁-R₁₂ can be —H, wherein R is defined as above. In someembodiments, dye compounds of the present teachings can comprise thestructure (II), wherein R₆ can be —H and R₇ can be —H, —SO₃H, —SO₃R,—CH₂CO₂H, —CH₂CO₂R, —CH₂NH₂, or —CH₂NHR, wherein R is defined as above,R₁₁ can be —CO₂H, —CO₂R, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H,—CH₂SO₃R, —CH₂NH₂, —CH₂NHR or —NO₂, and R₉-R₁₀ and R₁₂ can be —H,wherein R is defined as above. In some embodiments, dye compounds of thepresent teachings can comprise the structure (II), wherein R₆ can be —Hand R₇ can be —H, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂NH₂, or —CH₂NHR,wherein R is defined as above, R₁₂ can be —CO₂H, —CO₂R, —SO₃H, —SO₃R,—CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂, —CH₂NHR or —NO₂ andR₉-R₁₁ can be —H, wherein R is defined as above.

In some embodiments, any of R₉-R₁₂ can be —SO₃H, —SO₃R, —CH₂NH₂,—CH₂NHR, or —NO₂, wherein R is defined as above. In some embodiments,any of R₉-R₁₂ can be —SO₃H or —SO₃R, wherein R is defined as above. Insome embodiments, any of R₉-R₁₂ can be —CH₂NH₂ or —CH₂NHR, wherein R isdefined as above.

In some embodiments, a dye compound of the present teachings cancomprise the structure

wherein R₆ and R₇ can be hydrogen, fluorine, chlorine or bromine.

In some embodiments, a dye compound of the present teachings cancomprise the structure

wherein R₆ and R₇ can be hydrogen, fluorine, chlorine or bromine.

In some embodiments, a dye compound of the present teachings cancomprise the structure

wherein R₆ and R₇ can be hydrogen, fluorine, chlorine or bromine.

In some embodiments, a dye compound of the present teachings cancomprise the structure

wherein R₆ and R₇ can be hydrogen, fluorine, chlorine or bromine.

In some embodiments, a dye compound of the present teachings cancomprise the structure

In some embodiments, a dye compound of the present teachings cancomprise the structure

wherein R is defined as above.

In some embodiments, a dye compound of the present teachings cancomprise the structure

In some embodiments, a dye compound of the present teachings cancomprise the structure

wherein R is defined as above.

In some embodiments, R can be substituted benzoyl. In some embodiments,R can be linking group. In some embodiments, R can be trifluoroacetyl.In some embodiments, R can comprise the structure

In some embodiments, R can comprise the structure

X can be succinimide.

In some embodiments, R can be

In some embodiments, compounds of the present teachings comprise thestructure (III). In some embodiments, R₁ and R₃ can be —H. In someembodiments, R₆ can be —H. In some embodiments, R₁₃, R₁₅ and R₁₆ can be—H. In some embodiments, R₂ can be —SO₃H. In some embodiments, R₂ can be—H. In some embodiments, R₁₄ can be —CH₂NH₂. In some embodiments, R₁₄can be —CH₂NHR. In some embodiments, R can be

In some embodiments, R can comprise the structure

X can be succinimide.

In some embodiments, R can be

In some embodiments, R can be linking group. In some embodiments, R₁₄can be —H. In some embodiments, R₁₄ can be —SO₃H. In some embodiments,R₁₄ can be —H. In some embodiments, R₂ can be —CH₂NH₂. In someembodiments, R₂ can be —CH₂NHR. In some embodiments, R can be

In some embodiments, R can comprise the structure

X can be succinimide.

In some embodiments, R can be

In some embodiments, R can be linking group. In some embodiments, R₂ canbe —H. In some embodiments, R₄ and R₅ can be methyl.

In some embodiments, compounds of the present teachings comprise thestructure (IV). In some embodiments, R₁₀ can be —SO₃H. In someembodiments, R₁₀ can be —H. In some embodiments, R₁₄ can be —CH₂NH₂. Insome embodiments, R₁₄ can be —CH₂NHR. In some embodiments, R can be

In some embodiments, R can comprise the structure

X can be succinimide.

In some embodiments, R can be

In some embodiments, R is linking group. In some embodiments, R₁₄ can be—H. In some embodiments, R₃, R₆, R₉, R₁₁, R₁₂, R₁₃, R₁₅ and R₁₆ can be—H. In some embodiments, R₁₄ can be —SO₃H. In some embodiments, R₁₄ canbe —H. In some embodiments, R₁₀ can be —CH₂NH₂. In some embodiments, R₁₀can be —CH₂NHR. In some embodiments, R can be

In some embodiments, R can comprise the structure

X can be succinimide.

In some embodiments, R can be

In some embodiments, R can be linking group. In some embodiments, R₁₄can be —H. In some embodiments, R₃, R₆, R₉, R₁₁, R₁₂, R₁₃, R₁₅ and R₁₆can be —H. In some embodiments, R₄ and R₅ can be methyl.

As used herein “substituted” refers to a molecule wherein one or morehydrogen atoms are replaced with one or more non-hydrogen atoms,functional groups or moieties. For example, unsubstituted amine is —NH₂,while a substituted amine can be —NHCH₃. Exemplary substituents includebut are not limited to halogen, fluorine, chlorine, bromine, C₁-C₆alkyl, C₁-C₆ cycloalkyl, C₁-C₆ branched alkyl, C₁-C₆ alkene, C₁-C₆cyclic alkene, C₁-C₆ branched alkene, C₁-C₆ alkyne, C₁-C₆ branchedalkyne, sulfate, sulfonate, sulfone, amino, ammonium, amido, nitrile,C₁-C₆ alkoxy, phenoxy, substituted phenoxy aromatic, phenyl, polycyclicaromatic, electron-rich heterocycle, and linking group.

As used herein, “linking group” refers to a moiety capable of reactingwith a “complementary functionality” attached to a reagent or member ofan energy transfer dye pair, such reaction forming a “linkage”connecting the dye to the reagent or member of the energy transfer dyepair. Suitable linking groups include but are not limited toisothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinyl, succinimidylester, or other active carboxylate whenever the complementaryfunctionality is amine. Suitable linking groups include but are notlimited to maleimide, haloacetyl, iodoacetyl, haloacetamide oriodoacetamide whenever the complementary functionality is sulfhydryl.See, for example, R. Haugland, Molecular Probes Handbook of FluorescentProbes and Research Chemicals, Molecular probes, Inc. (1992).

Furthermore, it will be understood that a variety of complementarylinking group/complementary functionality pairs suitable for covalentlyconjugating dye molecules of the present teachings to various moleculesor substrates (i.e.—nucleotides, nucleosides, oligonucleotides,peptides, other dyes molecules, linking moieties, and the like) areknown in the art. Examples of complementary electrophiles andnucleophiles suitable for use as linking group/complementaryfunctionality pairs in a wide variety of contexts are shown in Table 1,where the reaction of the indicated electrophilic and nucleophilicspecies yields the indicated covalent linkage. Conditions under whichthe covalent linkages are formed are well-known.

TABLE 1 Examples Of Some Routes To Useful Covalent LinkagesElectrophilic Group Nucleophilic Group Resulting Linkage activatedesters* amines/anilines carboxamides acyl azides** amines/anilinescarboxamides acyl halides amines/anilines carboxamides acyl halidesalcohols/phenols esters acyl nitriles alcohols/phenols esters acylnitriles amines/anilines carboxamides aldehydes amines/anilines iminesaldehydes or ketones hydrazines hydrazones aldehydes or ketoneshydroxylamines oximes alkyl halides amines/anilines alkyl amines alkylhalides carboxylic acids esters alkyl halides thiols thioethers alkylhalides alcohols/phenols ethers alkyl sulfonates thiols thioethers alkylsulfonates carboxylic acids esters alkyl sulfonates alcohols/phenolsethers anhydrides alcohols/phenols esters anhydrides amines/anilinescarboxamides aryl halides thiols thiophenols aryl halides amines arylamines aziridines thiols thioethers boronates glycols boronate esterscarboxylic acids amines/anilines carboxamides carboxylic acids alcoholsesters carboxylic acids hydrazines hydrazides carbodiimides carboxylicacids N-acylureas or anhydrides carbonates amines carbamateschloroformates amine carbamates diazoalkanes carboxylic acids estersepoxides thiols thioethers haloacetamides thiols thioethershalotriazines amines/anilines aminotriazines halotriazinesalcohols/phenols triazinyl ethers imido esters amines/anilines amidinesisocyanates amines/anilines ureas isocyanates alcohols/phenols urethanesisothiocyanates amineslanilines thioureas maleimides thiols thioethersphosphoramidites alcohols phosphite esters silyl halides alcohols silylethers sulfonate esters amines/anilines alkyl amines sulfonate estersthiols thioethers sulfonate esters carboxylic acids esters sulfonateesters alcohols ethers sulfonyl halides amines/anilines sulfonamidessulfonyl halides phenols/alcohols sulfonate esters *Activated esters, asunderstood in the art, generally have the formula —COX, where X is agood leaving group, e.g., oxysuccinimidyl (—ONC₄H₄O₂),oxysulfosuccinimidyl (—ONC₄H₃O₂—SO₃H), 1-oxybenzotriazoiyl (—OC₆H₄N₃);or an aryloxy group of the formula —OR″, where R″ is an aryl or an arylsubstituted with one or more of the same or differentelectron-withdrawing substituents (e.g., —NO₂, —F, —Cl, —CN or —CF₃),used to form an anhydride or mixed anhydride of the formula —OCOR^(a) or—OCNR^(a)NHR^(b), where R^(a) and R^(b), which may be the same ordifferent, are (C₁-C₆) alkyl, (C₁-C₆) perfluoroalkyl or (C₁-C₆) alkoxy;or cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl. **Acylazides can also rearrange to isocyanates.

The selection of nucleophile or electrophile used to covalentlyconjugate a dye of the present teachings to a given molecule orsubstrate can depend upon the identity of the complementary functionalgroup on the molecule or substrate to which the dye molecule is to beconjugated. Types of complementary functional groups that can be presenton molecules or substances to be conjugated include, but are not limitedto, amines, thiols, alcohols, phenols, aldehydes, ketones, phosphates,imidazoles, hydrazines, hydroxylamines, mono- and disubstituted amines,halides, epoxides, sulfonate esters, carboxylic acids or carboxylates.

In some embodiments, suitable nucleophiles for use in connection withthe present teachings comprise amines, phenols, anilines, thiols oralcohols, or combinations thereof. In some embodiments, the nucleophilecomprises an amine. In some embodiments, the nucleophile comprises aprimary amine. In some embodiments, the nucleophile comprises asecondary amine.

In some embodiments, the electrophile comprises an acrylamide, anactivated ester of a carboxylic acid, an acyl azide, an acyl nitrile, anacyl halide, an aldehyde, an alkyl halide, an anhydride, an aryl halide,an azide, an aziridine, a boronate, a carboxylic acid or carboxylate, adiazoalkane, a haloacetamide, a halotriazine, a hydrazine, an imidoester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramiditea Michael acceptor (i.e.—an α,β-unsaturated ester, an α,β-unsaturatedaldehyde, and the like) or a sulfonyl halide.

In some embodiments, the electrophile comprises an activated ester of acarboxylic acid or carboxylate, a succinimidyl ester, a haloacetamide,an acyl halide, an alkyl halide, a sulfonyl halide, an isothiocyanate, amaleimide or an azidoperfluorobenzamido group. In some embodiments, thelinking group is a N-hydroxysuccinimidyl (NHS) ester and thecomplementary functionality is an amine. To form an NHS ester, a dye ofthe present teachings including a carboxylic acid moiety as a linkinggroup is reacted with, for example, dicyclohexylcarbodiimide (DCC) andN-hydroxysuccinimide. Alternatively, to form an NHS ester, a molecule orsubstrate to be conjugated to a dye molecule of the present teachingsincluding a carboxylic acid moiety as a linking group is reacted with,for example, dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide.

An exemplary synthetic scheme for the preparation of compounds of thepresent teachings comprising the structure (1) is shown in FIG. 1.Using, for example, the synthetic procedures described in Corey, P. F.,U.S. Pat. No. 4,810,636 as a guide, one of skill in the art can react a4-hydroxyaniline compound of the type (2) with a tertiary alcohol (4),such as 2-(4′-hydroxyphenyl)-2-propanol, in the presence of a base toform compound (5). Alternatively, one can oxidize 4-hydroxyanilinecompound (2) to form an N-chloroimine compound of the type (I) which canbe reacted with a tertiary alcohol (4), such as2-(4′-hydroxyphenyl)-2-propanol, in the presence of a base to formcompound (5). Compound (5) can then be reacted with a reducing agent,such as sodium dithionite, to form secondary amine compound (6).Compound (6) can then be converted to compound (7) by cyclization,through treatment with an acid such as 2N HCl, followed by oxidation,with for example sodium periodate. In some embodiments, depending on thesubstitution pattern present, compound (7) can be optionally be furtherderivitized. For example, compound (7) can optionally be sulfonated byreaction with, for example, chlorosulfonic acid.

Alternatively, compound (7) can be aminomethylated by reaction with anaminomethylating agent, for example, N-(hydroxymethyl)trifluoroacetamidein the presence of an acid such as concentrated sulfuric acid, orsimilar established conditions.

An exemplary synthetic scheme for the preparation of compounds of thepresent teachings comprising the structure (II) is shown in FIG. 2.Following a scheme similar to that described by Corey, a4-hydroxyaniline compound of the type (2) can be reacted with a tertiaryalcohol (8), such as 2-(4′-hydroxynathalen-2-yl)-2-propanol, in thepresence of a base to form compound (9). Alternatively, 4-hydroxyanilinecompound (2) can be oxidized using known conditions to form anN-chloroimine compound of the type (3) which can be reacted with atertiary alcohol (8) in the presence of a base to form compound (9).Compound (9) can then be reacted with a reducing agent, such as sodiumdithionite, to form secondary amine compound (10). Compound (10) canthen be converted to compound (11) by cyclization, through treatmentwith an acid such as 2N HCl, followed by oxidation, with for examplesodium periodate. In some embodiments, depending on the substitutionpattern present, compound (11) can be optionally be further derivitized.For example, compound (11) can optionally be sulfonated by reactionwith, for example, chlorosulfonic acid.

An exemplary synthetic scheme for the preparation of compounds of thepresent teachings comprising the structure (III) is shown in FIG. 3.Following a scheme similar to that described by Corey, a compound of thetype (12) can be reacted with a tertiary alcohol (4), in the presence ofa base to form compound (14). Alternatively, compound (12) can beoxidized using known conditions to form an N-chloroimine compound of thetype (13) which can be reacted with a tertiary alcohol (4) in thepresence of a base to form compound (14). Compound (14) can then bereacted with a reducing agent, such as sodium dithionite, to formsecondary amine compound (15). Compound (15 can then be converted tocompound (16) by cyclization, through treatment with an acid such as 2NHCl, followed by oxidation, with for example sodium periodate. In someembodiments, depending on the substitution pattern present, compound(16) can optionally be further derivitized. For example, compound (16)can optionally be sulfonated by reaction with, for example,chlorosulfonic acid.

An exemplary synthetic scheme for the preparation of compounds of thepresent teachings comprising the structure (IV) is shown in FIG. 4.Following a scheme similar to that described by Corey, a compound of thetype (12) can be reacted with a tertiary alcohol (8), in the presence ofa base to form compound (17). Alternatively, compound (12) can beoxidized using known conditions to form an N-chloroimine compound of thetype (13) which can be reacted with a tertiary alcohol (8) in thepresence of a base to form compound (17). Compound (17) can then bereacted with a reducing agent, such as sodium dithionite, to formsecondary amine compound (18). Compound (18) can then be converted tocompound (19) by cyclization, through treatment with an acid such as 2NHCl, followed by oxidation, with for example sodium periodate. In someembodiments, depending on the substitution pattern present, compound(19) can optionally be further derivitized. For example, compound (19)can optionally be sulfonated by reaction with, for example,chlorosulfonic acid.

A synthetic scheme for the preparation of exemplary compounds of thepresent teachings is shown in FIG. 5. Following a scheme similar to thatdescribed by Corey, a chloroimine compound of the type (20) can bereacted with a tertiary alcohol (21), in the presence of a base to formcompound (22). Compound (22) can then be reacted with a reducing agent,such as sodium dithionite, to form secondary amine compound (23).Compound (23) can then be converted to compound (24) by cyclization,through treatment with an acid such as 2N HCl, followed by oxidation,with for example sodium periodate. Further, compound (24) can besulfonated by reaction with, for example, chlorosulfonic acid to providedye (25) of the present teachings. Alternatively, compound (24) can beaminomethylated by reaction with an aminomethylating agent, for example,N-(hydroxymethyl)trifluoroacetamide in the presence of an acid such asconcentrated sulfuric acid, or similar established conditions to formcompound (26). Dye (26) can be de-halogenated by literature procedure(Corey, P. F., U.S. Pat. No. 4,810,636 Mar. 7, 1989; Corey, et al. AngewChem. Int. Ed. Engl 30 (1991)), by reduction with, for example, Raneynickel/H₂ and then oxidized by reacting with an oxidizing agent, such assodium periodate, to give dye (27) of the present teachings. Dye (27)can be can be sulfonated by reaction with, for example, chlorosulfonicacid to provide dye (28) of the present teachings.

If one of skill in the art were to use2-(4′-hydroxynathalen-2-yl)-2-propanol as the tertiary alcohol, it couldbe prepared according to the literature procedures (Haworth et al. J.Chem Soc, Abstracts, pp. 10-13 (1943)), see FIG. 6. Specifically, benzylsuccinate (29) (commercially available from Sigma-Aldrich ChemicalCompany, Milwaukee, Wis.) can be suspended in cold acetyl chloride togive a bis-anhydride benzyl succinate derivative. The anhydridederivative can be cyclized to the tetralone intermediate with AlCl₃ innitrobenzene. The tetralone intermediate can be aromatized in two stepsby bromination and base catalyzed bromide elimination to give4-hydroxy-2-napthoic acid (30). 4-Hydroxy-2-napthoic acid compound (30)can be converted to the ethyl ester derivative by Fisher esterificationin ethanol and HCl. Finally, using established literature procedures (J.Am. Chem. Soc., v. 108, 4119 (1986)), ethyl 4-Hydroxy-2-napthoate can bereacted with methyl magnesium chloride (3.3 equiv) to give the tertiaryalcohol 2-(4′-hydroxynathalen-2-yl)-2-propanol (31).

A synthetic scheme for the preparation of exemplary compounds of thepresent teachings is shown in FIG. 7. Following a scheme similar to thatdescribed by Corey, N-chloroimine compound (20) can be reacted withtertiary alcohol (31), in the presence of a base to form compound (32).Compound (32) can then be reacted with a reducing agent, such as sodiumdithionite, to form secondary amine compound (33). Compound (33) canthen be converted to compound (34) by cyclization, through treatmentwith an acid such as 2N HCl, followed by oxidation, with for examplesodium periodate.

A synthetic scheme for the conversion of exemplary compound (34) tofurther compounds of the present teachings is shown in FIG. 8. In someembodiments, dye (34) can be can be sulfonated by reaction with, forexample, chlorosulfonic acid to provide dye (35) of the presentteachings. Dye (35) can be de-halogenated by literature procedure(Corey, P. F., U.S. Pat. No. 4,810,636 Mar. 7, 1989; Corey, et al. AngewChem. Int. Ed. Engl. 30 (1991)), by reduction with, for example, Raneynickel/H₂ and then oxidized by reacting with an oxidizing agent, such assodium periodate, to give dye (36) of the present teachings. Dye (36)can be aminomethylated by treating withN-(hydroxymethyl)trifluoroacetamide in the presence of an acid such asconcentrated sulfuric acid, or similar established conditions to formcompound (37) of the present teachings. Alternatively, dye (34) can beaminomethylated by treating with N-(hydroxymethyl)trifluoroacetamide inthe presence of an acid such as concentrated sulfuric acid, or similarestablished conditions to form compound (38). As above, compound (38)can be de-halogenated and then oxidized by literature procedures to givecompound (39). Finally, compound (39) can be sulfonated by reactionwith, for example, chlorosulfonic acid to provide a dye (40) of thepresent teachings. Further, one of skill in the art will recognize thatthe amine functionality on compounds (37) or (40) can optionally bedeprotected and optionally converted to a substituted amine and/orcompounds (37) or (40) can optionally be halogenated using establishedprocedures to tune the fluorescence properties (i.e.—emissionwavelength).

Suitable N-chloroimine compounds of the type (3), shown in, for example,FIGS. 1 & 2, for use in connection with the present teachings can beobtained from commercial sources and/or prepared from numerous4-hydroxyaniline compounds that are either commercially available or areknown in the art using literature established procedures. Examples, ofsuitable 4-hydroxyaniline compounds include, but are not limited to, thefollowing:

One of skill in the art will understand that any of the above4-hydroxyaniline compounds can be converted into the N-chloroiminedirectly or after optional protection of possibly labile functionality.Further examples of suitable for 4-hydroxyaniline compounds that areknown in the art can be found by structure searching in availabledatabases such as Chemical Abstracts Service (CAS), SciFinder, and thelike.

Suitable N-chloroimine compounds of the type (12), shown in, forexample, FIG. 3, for use in connection with the present teachings can beobtained from commercial sources and/or prepared from numerous4-hydroxy-aminonaphthalene compounds that are either commerciallyavailable or are known in the art using literature establishedprocedures. Examples, of suitable 4-hydroxy-aminonaphthalene compoundsinclude, but are not limited to, the following:

One of skill in the art will understand that any of the above4-hydroxy-aminonaphthalene compounds can be converted into theN-chloroimine directly or after optional protection of possibly labilefunctionality. Further examples of suitable for4-hydroxy-aminonaphthalene compounds that are known in the art can befound by structure searching in available databases such as ChemicalAbstracts Service (CAS), SciFinder, and the like.

Suitable tertiary alcohol compounds of the type (8) shown in, forexample, FIG. 2, can be prepared from suitable 4-hydroxy-2-napthoic acidcompounds in a manner similar to that described in FIG. 6. Examples ofsuitable 4-hydroxy-2-napthoic acid compounds known in the art include,but are not limited to, the following:

One of skill in the art will understand that any of the above4-hydroxy-2-napthoic acid compounds can be used directly forsynthesizing compounds of the present teachings or after optionalprotection of possibly labile functionality. Further examples ofsuitable for 4-hydroxy-2-napthoic acid compounds that are known in theart can be found by structure searching in available databases such asChemical Abstracts Service (CAS), SciFinder, and the like.

In some embodiments, the present teachings comprise energy transfer dyecompounds incorporating dye compounds of Formula I-IV. Generally, energytransfer dyes of the present teachings comprise a donor dye capable ofabsorbing light at a first wavelength and emitting excitation energy inresponse that is covalently attached to an acceptor dye which is capableof absorbing the excitation energy emitted by the donor dye andfluorescing at a second wavelength in response. In some embodiments, thedonor dye can be covalently attached to the acceptor dye through alinker. In some embodiments, the linker can be effective to facilitateefficient energy transfer between the donor and acceptor dyes. In someembodiments, the linker can be non-nucleotidic. In some embodiments, thelinker can be a nucleotidic linker, such as a polynucleotide. For athorough discussion of the structure, synthesis and use of such energytransfer dyes see, for example, Mathies, et al. U.S. Pat. No. 5,728,528,Lee, et al. U.S. Pat. No. 5,863,727, Glazer, et al. U.S. Pat. No.5,853,992, Waggoner, et al., U.S. Pat. No. 6,008,373, Nampalli, et al.,U.S. Patent Application Pub. No. 2004/0126763 A1, Kumar, et al., PCTPub. No. WO 00/13026A1 and PCT Pub. No. WO 01/19841A1, each of which isincorporated herein by reference for all it discloses with regard toenergy transfer dye structures, energy transfer dye synthesis, energytransfer dye linkers, alternative donor dyes, alternative acceptor dyesand energy transfer dye spectral properties.

In some embodiments, linkers suitable for use in connection with thepresent teachings can comprise the general structure

wherein carbonyl can be covalently attached to either a donor dye or anacceptor dye, R₁₁ can be a moiety that comprises an unsubstitutedalkene, a substituted alkene, an unsubstituted diene, a substituteddiene, an unsubstituted triene, a substituted triene, an unsubstitutedalkyne, a substituted alkyne, an unsubstituted five- or six-memberedring having at least one unsaturated bond, a substituted five- orsix-membered ring having at least one unsaturated bond or anunsubstituted or substituted fused ring structure that is attached tothe carbonyl carbon atom, and R₁₂ is a moiety comprising a functionalgroup that is capable of attaching the linker to a donor dye or anacceptor dye, such that both a donor dye and an acceptor dye arerepresented.

Examples of suitable five- or six-membered rings that can be used as R₁₁in the linker include, but are not limited to cyclopentene, cyclohexene,cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, pyrazole,isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine,triazine, pyrazine and oxazine. Examples of fused ring structuresinclude, but are not limited to indene, benzofuran, thionaphthalene,indole and naphthalene. In some embodiments, the linker has thestructure

In some embodiments, the linker attaches to a dye of the presentteachings at one of the X₁-X₂, R₁-R₃ or R₇-R₁₀ positions. In someembodiments, the linker can be a bond. Additional suitable linkersinclude polynucleotides, ribonucleic acids, and the like.

In some embodiments, one of the donor or acceptor dye is a dye accordingto the present teachings and the other dye can be a cyanine,phthalocyanine, squaraine, bodipy, fluorescein, rhodamine, extendedrhodamine or dibenzorhodamine dye.

Example of suitable dyes for use in connection with energy transfer dyesof the present teachings include, but are not limited to,5-carboxyfluorescein, 6-carboxyfluorescein, rhodamine green (R110),5-carboxyrhodamine, 6-carboxyrhodamine,N,N′-diethyl-2′,7′-dimethyl-5-carboxy-rhodamine (5-R6G),N,N′-diethyl-2′,7′-dimethyl-6-carboxyrhodamine (6-R6G),N,N,N′,N′-tetramethyl-5-carboxyrhodamine (5-TAMRA),N,N,N′,N′-tetramethyl-5-carboxyrhodamine (6-TAMRA),5-carboxy-X-rhodamine (5-ROX), 6-carboxy-X-rhodamine (6-ROX),5-carboxy-2′,4′,5′,7′,-4,7-hexachlorofluorescein,6-carboxy-2′,4′,5′,7′,4,7-hexachloro-fluorescein,5-carboxy-2′,7′-dicarboxy-4′,5′-dichlorofluorescein,6-carboxy-2′,7′-dicarboxy-4′,5′-dichloro-fluorescein,5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,1′,2′-benzo-4′-fluoro-7′,4,7-trichloro-5-carboxyfluorescein,1′,2′-benzo-4′-fluoro-7′,4,7-trichloro-6-carboxy-fluorescein,1′,2′,7′,8′-dibenzo-4,7-dichloro-5-carboxyfluorescein, as well as othercommercially available dyes as shown in Table 2.

TABLE 2 Absorbance Emission Extinction Fluorescent Dye (nm) (nm)Coefficient 5-Fluorescein 495 520 73000 5-Carboxyfluorescein (5-FAM) 495520 83000 6-Carboxyfluorescein (6-FAM) 495 520 830006-Carboxyhexachlorofluorescein 535 556 73000 (6-HEX)6-Carboxytetrachlorofluorescein 521 536 73000 (6-TET) JOE 520 548 73000LightCycler Red 640 625 640 LightCycler Red 705 685 705 Oregon Green 488496 516 76000 Oregon Green 500 499 519 84000 Oregon Green 514 506 52685000 BODIPY FL-X 504 510 70000 BODIPY FL 504 510 70000 BODIPY-TMR-X 544570 56000 BODIPY R6G 528 547 70000 BODIPY 650/665 650 665 101000 BODIPY564/570 563 569 142000 BODIPY 581/591 581 591 136000 BODIPY TR-X 588 61668000 BODIPY 630/650 625 640 101000 BODIPY 493/503 500 509 790005-Carboxyrhodamine 6G 524 557 102000 5(6)-Carboxytetramethylrhodamine546 576 90000 (TAMRA) 6-Carboxytetramethylrhodamine 544 576 90000(TAMRA) 5(6)-Carboxy-X-Rhodamine (ROX) 576 601 820006-Carboxy-X-Rhodamine (ROX) 575 602 82000 AMCA-X (Coumarin) 353 44219000 Texas Red-X 583 603 116000 Rhodamine Red-X 560 580 129000 MarinaBlue 362 459 19000 Pacific Blue 416 451 37000 Rhodamine Green-X 503 52874000 7-diethylaminocoumarin-3- 432 472 56000 carboxylic acid7-methoxycoumarin-3-carboxylic 358 410 26000 acid Cy3 552 570 150000Cy3B 558 573 130000 Cy5 643 667 250000 Cy5.5 675 694 250000 DY-505 505530 85000 DY-550 553 578 122000 DY-555 555 580 100000 DY-610 606 636140000 DY-630 630 655 120000 DY-633 630 659 120000 DY-636 645 671 120000DY-650 653 674 77000 DY-675 674 699 110000 DY-676 674 699 84000 DY-681691 708 125000 DY-700 702 723 96000 DY-701 706 731 115000 DY-730 734 750113000 DY-750 747 776 45700 DY-751 751 779 220000 DY-782 782 800 102000Cy3.5 581 596 150000 EDANS 336 490 5700 WellRED D2-PA 750 770 170000WellRED D3-PA 685 706 224000 WellRED D4-PA 650 670 203000 Pyrene 341 37743000 Cascade Blue 399 423 30000 Cascade Yellow 409 558 24000 PyMPO 415570 26000 Lucifer Yellow 428 532 11000 NBD-X 466 535 22000Carboxynapthofluorescein 598 668 42000 Alexa Fluor 350 346 442 19000Alexa Fluor 405 401 421 35000 Alexa Fluor 430 434 541 16000 Alexa Fluor488 495 519 71000 Alexa Fluor 532 532 554 81000 Alexa Fluor 546 556 573104000 Alexa Fluor 555 555 565 150000 Alexa Fluor 568 578 603 91300Alexa Fluor 594 590 617 73000 Alexa Fluor 633 632 647 100000 Alexa Fluor647 650 665 239000 Alexa Fluor 660 663 690 132000 Alexa Fluor 680 679702 184000 Alexa Fluor 700 702 723 192000 Alexa Fluor 750 749 775 240000Oyster 556 556 570 155000 Oyster 645 645 666 250000 Oyster 656 656 674220000 5(6)-Carboxyeosin 521 544 95000 Erythrosin 529 544 90000

In some embodiments, the present teachings provide for labelednucleosides and/or nucleotides comprising the structureNUC-L-D

wherein NUC comprises a nucleoside, a nucleotide, a modified nucleosideor a modified nucleotide, L comprises a bond or a linker and D comprisesa dye compound of the present teachings. In some embodiments, NUC and Dcan be conjugated by a linker, L, wherein L can be attached to D at oneof X₁-X₂, R₁-R₃ or R₇-R₁₀. In some embodiments, if NUC comprises apurine base, the linker can be attached to the 8-position of the purine,if NUC comprises a 7-deazapurine base, the linker can be attached to the7-position of the 7-deazapurine, and if NUC comprises a pyrimidine base,the linker can be attached to the 5-position of the pyrimidine. Suchnucleoside and nucleotide reagents can be particularly useful in thecontext of labeling polynucleotides formed by enzymatic synthesis, e.g.,nucleotide triphosphates used in the context of PCR amplification,Sanger-type polynucleotide sequencing, and nick-translation reactions.

It will be understood that nucleoside labeling can be accomplished byany of number of known labeling techniques employing known linkers,linking groups, and associated complementary functionalities. Generally,the linker should (i) not interfere with oligonucleotide-targethybridization, (ii) be compatible with relevant enzymes, e.g.,polymerases, ligases, and the like, and (iii) not adversely affect thefluorescence properties of the dye. For exemplary base labelingprocedures suitable for use in connection with the present teachingssee, for example, Gibson, et al., Nucleic Acids Research, 15:6455-6467(1987); Gebeyehu, et al., Nucleic Acids Research, 15: 4513-4535 (1987);Haralambidis, et al., Nucleic Acids Research, 15: 4856-4876 (1987);Nelson, et al., Nucleosides and Nucleotides, 5(3): 233-241 (1986);Bergstrom, et al., JACS, 111: 374-375 (1989); and U.S. Pat. Nos.4,855,225, 5,231,191, and 5,449,767.

In some embodiments, suitable linkers can be acetylenic amido or alkenicamido linkers, wherein the conjugation between the dye and thenucleoside or nucleotide base can be formed by, for example, reaction ofan activated N-hydroxysuccinimide (NHS) ester of the dye with analkynylamino- or alkenylamino-derivatized base of a nucleoside ornucleotide. In some embodiments, labeled nucleosides or nucleotides cancomprise the structure

In some embodiments, labeled nucleosides or nucleotides can comprise thestructure

wherein X can be

where n ranges from 1 to 5,

where n ranges from 1 to 5,

wherein R₁ can be —H or lower alkyl; and R₂ can be —H, lower alkyl orprotecting group. See, for example, Khan et al., U.S. patent applicationSer. No. 08/833,854 filed Apr. 10, 1997.

The synthesis of alkynylamino-derivatized nucleosides is taught by, forexample, Hobbs, et al. in European Patent No. 0 251 786 B1, and Hobbs,et al., J. Org. Chem., 54: 3420 (1989). Briefly, thealkynylamino-derivatized nucleotides can be formed by placing theappropriate halodideoxynucleoside (usually 5-iodopyrimidine and7-iodo-7-deazapurine dideoxynucleosides as taught by Hobbs, et al.(cited above)) and Cu(I) in a flask, flushing with argon to remove air,adding dry DMF, followed by addition of an alkynylamine, triethylamineand Pd(0). The reaction mixture is stirred for several hours, or untilthin layer chromatography indicates consumption of thehalodideoxynucleoside. When an unprotected alkynylamine is used, thealkynylamino-nucleoside can be isolated by concentrating the reactionmixture and purifying by silica gel chromatography with an elutingsolvent that contains ammonium hydroxide to neutralize hydrohalidegenerated in the coupling reaction. When a protected alkynylamine isused, methanol/methylene chloride can be added to the reaction mixture,followed by the bicarbonate form of a strongly basic anion exchangeresin. The slurry can then be stirred for about 45 minutes, filtered,and the resin rinsed with additional methanol/methylene chloride. Thecombined filtrates can be concentrated and purified byflash-chromatography on silica gel using a methanol-methylene chloridegradient. The triphosphates are obtained by standard techniques.

In some embodiments, nucleosides and/or nucleotides of the presentteachings can comprise natural sugars (i.e.—ribose, 2′-deoxyribose, andthe like) or sugar analogues. As used herein, the term “sugar analog”refers to analogs of the sugar ribose. Exemplary ribose sugar analogsinclude, but are not limited to, substituted or unsubstituted furanoseshaving more or fewer than 5 ring atoms, e.g., erythroses and hexoses andsubstituted or unsubstituted 3-6 carbon acyclic sugars. Typicalsubstituted furanoses and acyclic sugars are those in which one or moreof the carbon atoms are substituted with one or more of the same ordifferent —R, —OR, —NRR or halogen groups, where each R is independently—H, (C₁-C₆)alkyl or (C₁-C₁₄)aryl. Examples of substituted furanoseshaving 5 ring atoms include but are not limited to 2′-deoxyribose,2′-(C₁-C₆)alkylribose, 2′-(C₁-C₆)alkoxyribose, 2′-(C₅-C₁₄)aryloxyribose,2′,3′-dideoxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose,2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose,2′-deoxy-3′-aminoribose, 2′-deoxy-3′-(C₁-C₆)alkylribose,2′-deoxy-3′-(C₁-C₆)alkoxyribose, 2′-deoxy-3′-(C₅-C₁₄)aryloxyribose,3′-(C₁-C₆)alkylribose-5′-triphosphate,2′-deoxy-3′-(C₁-C₆)alkylribose-5′-triphosphate,2′-deoxy-3′-(C₁-C₆)alkoxyribose-5′-triphosphate,2′-deoxy-3′-(C₅-C₁₄)aryloxyribose-5′-triphosphate,2′-deoxy-3′-haloribose-5′-triphosphate,2′-deoxy-3′-aminoribose-5′-triphosphate,2′,3′-dideoxyribose-5′-triphosphate or2′,3′-didehydroribose-5′-triphosphate. Further sugar analogs alsoinclude so called locked nucleic acids (LNAs) having the structure

and those described in Wengel, et al. WO 99/14226, incorporated hereinby reference.

In some embodiments, nucleosides and/or nucleotides of the presentteachings can have the structure

wherein B comprises a nucleoside or nucleotide base, such as uracil,cytosine, deazaadenine, or deazaguanosine; W₁ and W₂ taken separatelycan be —OH or a group capable of blocking polymerase-mediatedtemplate-directed polymerzation, e.g., —H, fluorine, and the like; W₃can be OH, or mono-, di- or triphosphate or a phosphate analog; and D isa dye compound of the present teachings. In some embodiments,nucleotides of the present teachings can be dideoxynucleotidetriphosphates having the structure including associated counterions ifpresent.

including associated counterions if present.

Labeled dideoxy nucleotides such as that shown above find particularapplication as chain terminating agents in Sanger-type DNA sequencingmethods utilizing fluorescent detection.

In some embodiments, nucleotides of the present teachings can bedeoxynucleotide triphosphates having the structure

including associated counterions if present.

Labeled deoxynucleotides such as that shown above find particularapplication as reagents for labeling polymerase extension products,e.g., in the polymerase chain reaction or nick-translation.

In some embodiments, the present teachings can provide polynucleotideslabeled with at least one dye of the present teachings. Such labeledpolynucleotides are useful in a number of important contexts includingas DNA sequencing primers, PCR primers, oligonucleotide hybridizationprobes, oligonucleotide ligation probes, and the like.

In some embodiments, labeled polynucleotides of the present teachingscan include multiple dyes located such that fluorescence energy transfertakes place between a donor dye and an acceptor dye. Such multi-dyeenergy-transfer polynucleotides find application as spectrally-tunablesequencing primers, see for example, Ju, et al., Proc. Natl. Acad. Sci.USA 92: 4347-4351 (1995), or as hybridization probes, see for example,Lee, et al. Nucleic Acids Research, 21: 3761-3766 (1993).

Labeled polynucleotides can be synthesized either enzymatically, e.g.,using a DNA polymerase or ligase, see for example, Stryer, Biochemistry,Chapter 24, W.H. Freeman and Company (1981), or by chemical synthesis,e.g., by the phosphoramidite method, the phosphitetriester method, andthe like, see for example, Gait, Oligonucleotide Synthesis, IRL Press(1990). Labels may be introduced during enzymatic synthesis utilizinglabeled nucleotide triphosphate monomers as described above, orintroduced during chemical synthesis using labeled non-nucleotide ornucleotide phosphoramidites as described above, or may be introducedsubsequent to synthesis.

Generally, if the labeled polynucleotide is made using enzymaticsynthesis, the following procedure can be used. A template DNA isdenatured and an oligonucleotide primer is annealed to the template DNA.A mixture of deoxynucleotide triphosphates is added to the mixtureincluding dGTP, dATP, dCTP, and dTTP where at least a fraction of thedeoxynucleotides is labeled with a dye compound of the invention asdescribed above. Next, a polymerase enzyme is added under conditionswhere the polymerase enzyme is active. A labeled polynucleotide isformed by the incorporation of the labeled deoxynucleotides duringpolymerase-mediated strand synthesis. In an alternative enzymaticsynthesis method, two primers are used instead of one, one primercomplementary to the (+) strand and the other complementary to the (−)strand of the target, the polymerase is a thermostable polymerase, andthe reaction temperature is cycled between a denaturation temperatureand an extension temperature, thereby exponentially synthesizing alabeled complement to the target sequence by PCR, see for example, PCRProtocols, Innis et al. eds., Academic Press (1990).

Labeled polynucleotides can be chemically synthesized using thephosphoramidite method. Detailed descriptions of the chemistry used toform polynucleotides by the phosphoramidite method are provided in, forexample, Caruthers et al., U.S. Pat. Nos. 4,458,066 and 4,415,732;Caruthers, et al., Genetic Engineering, 4: 1-17 (1982); Users ManualModel 392 and 394 Polynucleotide Synthesizers, pages 6-1 through 6-22,Applied Biosystems, Part No. 901237 (1991).

The phosphoramidite method of polynucleotide synthesis can beadvantageous in some embodiments because of its efficient and rapidcoupling reactions and the stability of the starting materials. Thesynthesis is performed with the growing polynucleotide chain attached toa solid support, so that excess reagents, which are in the liquid phase,can be easily removed by filtration, thereby eliminating the need forpurification steps between synthesis cycles.

The following briefly describes the steps of a typical polynucleotidesynthesis cycle using the phosphoramidite method. First, a solid supportincluding a protected nucleotide monomer is treated with acid, e.g.,trichloroacetic acid, to remove a 5′-hydroxyl protecting group, freeingthe hydroxyl for a subsequent coupling reaction. An activatedintermediate is then formed by simultaneously adding a protectedphosphoramidite nucleoside monomer and a weak acid, e.g., tetrazole, tothe reaction. The weak acid protonates the nitrogen of thephosphoramidite forming a reactive intermediate. Nucleoside addition iscomplete within 30 s. Next, a capping step is performed that terminatesany polynucleotide chains that did not undergo nucleoside addition.Capping can be accomplished with acetic anhydride and 1-methylimidazole.The internucleotide linkage is then converted from the phosphite to themore stable phosphotriester by oxidation using iodine as the preferredoxidizing agent and water as the oxygen donor. After oxidation, thehydroxyl protecting group is removed with a protic acid, such astrichloroacetic acid or dichloroacetic acid, and the cycle is repeateduntil chain elongation is complete. After synthesis, the polynucleotidechain is cleaved from the support using a base, such as ammoniumhydroxide or t-butyl amine. The cleavage reaction also removes anyphosphate protecting groups, such as cyanoethyl. Finally, the protectinggroups on the exocyclic amines of the bases and the hydroxyl protectinggroups on the dyes are removed by treating the polynucleotide solutionin base at an elevated temperature, e.g., 55° C. One of skill in the artwill recognize that a variety if reagents can be used to perform thesynthesis described above, and in some cases reagents are available tocarry out more than one step in a single reaction.

Any of the phosphoramidite nucleoside monomers may be dye-labeledphosphoramidites as described above. If the 5′-terminal position of thenucleotide is labeled, a labeled non-nucleotidic phosphoramidite of theinvention may be used during the final condensation step. If an internalposition of the oligonucleotide is labeled, a labeled nucleotidicphosphoramidite of the invention may be used during any of thecondensation steps.

Subsequent to synthesis, the polynucleotide may be labeled at a numberof positions including the 5′-terminus, see for example Oligonucleotidesand Analogs, Eckstein ed., Chapter 8, IRL Press (1991) and Orgel, etal., Nucleic Acids Research 11(18): 6513 (1983); U.S. Pat. No.5,118,800; the phosphodiester backbone, see for example Oligonucleotidesand Analogs, Eckstein ed., Chapter 9, IRL Press (1991); or at the3′-terminus, see for example Nelson, Nucleic Acids Research 20(23):6253-6259, and U.S. Pat. Nos. 5,401,837 and 5,141,813. For a throughreview of oligonucleotide labeling procedures see R. Haugland in ExcitedStates of Biopolymers, Steiner ed., Plenum Press, NY (1983).

In one post-synthesis chemical labeling method an oligonucleotide can belabeled as follows. A dye including a carboxy linking group is convertedto the N-hydroxysuccinimide ester by reacting with approximately 1equivalent of 1,3-dicyclohexylcarbodiimide and approximately 3equivalents of N-hydroxysuccinimide in dry ethyl acetate for 3 hours atroom temperature. The reaction mixture is washed with 5% HCl, dried overmagnesium sulfate, filtered, and concentrated to a solid which isresuspended in DMSO. The DMSO dye stock is then added in excess (10-20×)to an aminohexyl derivatized oligonucleotide in 0.25 Mbicarbonate/carbonate buffer at pH 9.4 and allowed to react for 6 hours,e.g., U.S. Pat. No. 4,757,141. The dye labeled oligonucleotide isseparated from unreacted dye by passage through a size-exclusionchromatography column eluting with buffer, e.g., 0.1 molar triethylamineacetate (TEAA). The fraction containing the crude labeledoligonucleotide is further purified by reverse phase HPLC employinggradient elution.

It will be understood that the following examples are meant to be merelyillustrative and are not meant to be limiting of the present teachingsin any way. Although the above description will be adequate to teach oneof skill in the art how to practice the present teachings, the followingexamples are provided as further guidance to those of skill in the art.

In some embodiments, the present teachings provide for a mixturecomprising at least one compound of the present teachings in any of theforms described herein and at least one other component comprising afluorescent dye. For example, the present teachings can provide for amixture of polynucleotides, wherein at least one polynucleotide of themixture comprises a compound of the present teachings and at least oneother polynucleotide comprising a fluorescent dye. Mixture componentscomprising fluorescent dyes of the present teachings can be prepared byany of the methods described herein. In some embodiments, the presentteachings provide for kits comprising at least one compound of thepresent teachings in any of the forms described herein.

EXAMPLES Materials and Methods

Unless otherwise indicated, all reagents were obtained fromSigma-Aldrich (Milwaukee, Wis.) and used as received from thedistributor. DDAO (1) was prepared as described in Corey, P. F., U.S.Pat. No. 4,810,636. NMR spectra were obtained using a Bruker 400 MHzAvance-NMR Spectrometer. Mass spec. data was obtained using an AppliedBiosystems API 1500 Mass Spectrometer. Fluorescence data was obtainedusing a Perkin Elmer LS-50B Luminescence Spectrophotometer. UV/Vis datawas obtained using a Hewlett Packard 8451A Diode ArraySpectrophotometer.

Example 1: Synthesis of 6-sulfo-DDAO (42)

To a stirred solution of 50 mg of DDAO (41) dissolved in 5 mLdichlorormethane (DCM) and cooled to 0° C. was added 0.5 mLchlorosulfonic acid. The reaction was stirred at 60° C. overnight andthen poured into ice-water. Unreacted starting material was extractedwith ethyl acetate (EtOAc) and then the product was extracted withn-butanol. The solvent was removed in vacuo to obtain 45 mg of6-sulfo-DDAO (42) as dark shiny crystals. ¹H NMR (CD₃OD): δ 7.9 (s, 1H),7.75 (s, 1H), 7.2 (s, 1H), 1.78 (s, 6H). MS: M+H=388.

Example 2: Synthesis of N-(4-caboxybenzoyl)-aminomethyl DDAO (45)

To a stirred solution of 450 mg DDAO (1.466 mmoles) in 10 mL of conc.sulfuric acid (H₂SO₄) was added 419 mg ofN-(hydroxymethyl)trifluoroacetamide (2.929 mmoles) at room temperature.The resulting mixture was stirred at room temperature for 2 hours. Thereaction was poured into 100 mL of ice water and extracted with 4×100 mLof 10% methanol/dichloromethane (MeOH/DCM). The organic layer was driedover sodium sulfate (Na₂SO₄), filtered and evaporated to dryness underreduced pressure. The residue was purified by silica gel chromatographyusing a gradient of 2:20:78 to 8:20:72 MeOH/ethyl acetate (EtOAc)/DCM)to give 456 mg of compound 43 as a reddish solid. ¹H NMR (DMSO-d₆): δ11.4 (br s, 1H, OH), 9.95 (t, 1H, NH), 7.82 (s, 1H), 7.34 (s, 1H), 7.03(s, 1H), 4.35 (d, 2H), 1.78 (s, 6H). MS: (ESI) M−H=431.2.

A solution of 64 mg of compound 43 (0.148 mmoles) in 30 mL NH₃/MeOH wasstirred at room temperature for 20 hours. The reaction was concentratedto dryness by evaporation and co-evaporation with MeOH. The solid wasrecrystallized from MeOH/DCM to give compound 44.

To a stirred solution of compound 44 in 6 mL dimethyl-formamide(DMF)/DCM (1:1) was added 118 mg of 4-carboxyethy terephthaloyl chloride(0.594 mmoles) and 0.2 mL diisopropylethylamine at room temperature. Theresulting mixture was allowed to stir at room temperature for 3 hours.The reaction was then quenched by addition of 10 mL sat. aq. sodiumbicarbonate (NaHCO₃) and 0.5 mL of a solution of sodium methoxide/MeOH(25%). The reaction mixture was then diluted with 20 mL H₂O andextracted 1×50 mL EtOAc. The organic layer was dried over Na₂SO₄,filtered and evaporated under reduced pressure. The residue was thentreated with a solution of lithium hydroxide (LiOH)/MeOH/H₂O (177 mg/20mL/5 mL) at 80° C. for 45 minutes. The reaction was then cooled to roomtemperature, evaporated under reduced pressure, re-dissolved in 30 mLH₂O, acidified with 1.5 mL 10% HCl, and extracted with 100 mL EtOAc. Theorganic layer was dried over Na₂SO₄, filtered and evaporated to drynessunder reduced pressure. The residue was purified by silica gelchromatography (using a gradient of 1:9 to 8:2 MeOH/DCM) to give 52 mgof compound 45 as a solid. ¹H NMR (DMSO-d₆): δ 8.84 (t, 1H, NH), 8.00(d, 2H), 7.34 (m, 3H), 6.97 (s, 1H), 6.52 (s, 1H), 4.24 (d, 2H), 1.70(s, 6H). MS: (ESI) M+H=485.0.

Example 3: Synthesis of 6,8-Dinitro-DDAO (46)

A solution of 50 mg of DDAO dissolved in 2 mL of a 1:1 solution ofH₂SO₄/HNO₃ was stirred overnight at room temperature. The reaction wasextracted with EtOAc. The solvent was removed in vacuo and the crudeproduct was recrystallized from EtOH to give 41 mg of dinitro-DDAO (46).

Example 4: Synthesis of N-(4-caboxy-2-sulfobenzoyl)-aminomethyl DDAO(47)

Compound 43 was prepared in the same manner as in Example 2. A solutionof 43.3 mg of compound 43 (0.1 mmoles) in 20 mL NH₃/MeOH was stirred atroom temperature for 20 hours. The reaction was concentrated to drynessby evaporation and co-evaporation with DCM to give compound 44.

The residue (44) was dissolved in 5 mL DMF and treated with 200 μL ofdiisopropylethylamine (Hunig's base) and 74 mg of anhydride 48 (0.324mmoles) at room temperature. The reaction was stirred at roomtemperature for 3 hours then quenched with 1 mL 10% HCl. The solvent wasremoved in vacuo and the residue was redissolved in 50 mL H₂O andextracted with 3×50 mL 10% MeOH/EtOAc. The organic layer was dried overNa₂SO₄, filtered and evaporated to dryness.

The residue was then treated with 20 mL of a solution ofLiOH.H₂O/MeOH/H₂O (0.85 g/100 mL/20 mL) at room temperature for 10minutes. The mixture was acidified with 1 mL 10% HCl and diluted with 50mL sat. NaCl. The mixture was extracted 2×100 mL 10% MeOH/EtOAc, thenthe organic layer was treated with 0.5 mL triethylamine, evaporated todryness and finally co-evaporated with MeOH. The crude product wasdissolved in 50 mL of 20% MeOH/DCM and purified by silica gelchromatography (2×17.5 cm, eluant gradient: 20%, 30%, 50%, 60%, 70% and80% MeOH/DCM (100 mL each gradient step, collecting 20 mL fractions)) togive 60 mg of compound 47. ¹H NMR (in MeOD): δ 8.52 (d, 1H), 8.01 (dd,1H), 7.71 (d, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.69 (s, 1H), 4.45 (s,2H, CH₂), 1.82 (s, 6H, 2×CH₃). MS (ESI) m/e 563.2 (calcd. forM−H=563.0).

The invention claimed is:
 1. A composition comprising: a) anoligonucleotide comprising a fluorescent dye; and b) an internalstandard dye compound; wherein the fluorescent dye of theoligonucleotide and the internal standard dye compound are spectrallyresolvable and further wherein the internal standard dye compound isselected from:

wherein R₁-R₃ and R₆-R₁₆ are each independently selected from —H,halogen, aryl, substituted aryl, heteroaryl, —CO₂H, —CO₂R, —SO₃H, —SO₃R,—CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂, —CH₂NHR, —NO₂, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy,C₁-C₆ alkoxyaryl, substituted C₁-C₆ alkoxyaryl, biphenyl, substitutedbiphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl,wherein R is selected from C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆alkoxy, substituted C₁-C₆ alkoxy, C₁-C₆ alkoxyaryl, substituted C₁-C₆alkoxyaryl, phenyl, substituted phenyl, biphenyl, substituted biphenyl,benzyl, substituted benzyl, benzoyl, and substituted benzoyl; and R₄ andR₅ taken separately are selected from C₁-C₆ alkyl and C₁-C₆ substitutedalkyl, R₄ and R₅ taken together are selected from C₃-C₇ cycloalkyl,C₄-C₇ unsaturated cycloalkyl, C₃-C₇ substituted cycloalkyl and C₄-C₇substituted unsaturated cycloalkyl; with the proviso that if theinternal standard dye compound is selected from compounds of formula(I), then at least one of R₁, R₂, R₃ or R₈ is not —H.
 2. The compositionof claim 1, wherein the internal standard dye compound is selected fromcompounds of formula (I).
 3. The composition of claim 1, wherein R₆ andR₇ are each independently halogens selected from fluorine, chlorine, andbromine.
 4. The composition of claim 1, wherein R₆ and R₇ are fluorineor R₆ and R₇ are chlorine.
 5. The composition of claim 1, wherein theinternal dye standard compound is selected from compounds of formula (I)and R₂ is selected from —SO₃H, —SO₃R, —CH₂NH₂, —CH₂NHR, and —NO₂.
 6. Akit comprising: a) an oligonucleotide labeled with a fluorescent dye;and b) an internal standard dye compound; wherein the fluorescent dye ofthe oligonucleotide and the internal standard dye compound arespectrally resolvable and further wherein the internal standard dyecompound is selected from:

wherein R₁-R₃ and R₆-R₁₆ are each independently selected from —H,halogen, aryl, substituted aryl, heteroaryl, —CO₂H, —CO₂R, —SO₃H, —SO₃R,—CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂, —CH₂NHR, —NO₂, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy,C₁-C₆ alkoxyaryl, substituted C₁-C₆ alkoxyaryl, biphenyl, substitutedbiphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl,wherein R is selected from C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆alkoxy, substituted C₁-C₆ alkoxy, C₁-C₆ alkoxyaryl, substituted C₁-C₆alkoxyaryl, phenyl, substituted phenyl, biphenyl, substituted biphenyl,benzyl, substituted benzyl, benzoyl, and substituted benzoyl; and R₄ andR₅ taken separately are selected from C₁-C₆ alkyl and C₁-C₆ substitutedalkyl, R₄ and R₅ taken together are selected from C₃-C₇ cycloalkyl,C₄-C₇ unsaturated cycloalkyl, C₃-C₇ substituted cycloalkyl and C₄-C₇substituted unsaturated cycloalkyl; with the proviso that if theinternal standard dye compound is selected from compounds of formula(I), then at least one of R₁, R₂, R₃ or R₈ is not —H.
 7. The kit ofclaim 6, wherein the internal standard dye compound is selected fromcompounds having a structure of formula (I).
 8. The kit of claim 7,wherein R₆ and R₇ are each independently halogens selected fromfluorine, chlorine, and bromine.
 9. The kit of claim 8, wherein R₆ andR₇ are fluorine or R₆ and R₇ are chlorine.
 10. The kit of claim 9,wherein the internal dye standard compound is selected from compounds offormula (I) and R₂ is selected from —SO₃H, —SO₃R, —CH₂NH₂, —CH₂NHR, and—NO₂.
 11. A composition comprising: a) an oligonucleotide comprising afluorescent dye; and b) an internal standard dye compound; wherein thefluorescent dye of the oligonucleotide and the internal standard dyecompound are spectrally resolvable and further wherein the internalstandard dye compound is:

wherein R₁-R₃ and R₆-R₈ are each independently selected from —H,halogen, aryl, substituted aryl, heteroaryl, —CO₂H, —CO₂R, —SO₃H, —SO₃R,—CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂, —CH₂NHR, —NO₂, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy,C₁-C₆ alkoxyaryl, substituted C₁-C₆ alkoxyaryl, biphenyl, substitutedbiphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl,wherein R is selected from C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆alkoxy, substituted C₁-C₆ alkoxy, C₁-C₆ alkoxyaryl, substituted C₁-C₆alkoxyaryl, phenyl, substituted phenyl, biphenyl, substituted biphenyl,benzyl, substituted benzyl, benzoyl, and substituted benzoyl; and R₄ andR₅ taken separately are selected from C₁-C₆ alkyl and C₁-C₆ substitutedalkyl, R₄ and R₅ taken together are selected from C₃-C₇ cycloalkyl,C₄-C₇ unsaturated cycloalkyl, C₃-C₇ substituted cycloalkyl and C₄-C₇substituted unsaturated cycloalkyl; with the proviso that at least oneof R₁, R₂, R₃ or R₈ is not —H.
 12. The composition of claim 11, whereinR₆ and R₇ are each independently halogens selected from fluorine,chlorine, and bromine.
 13. The composition of claim 12, wherein R₆ andR₇ are fluorine or R₆ and R₇ are chlorine.
 14. The composition of claim11, wherein R₂ is selected from —SO₃H, —SO₃R, —CH₂NH₂, —CH₂NHR, and—NO₂.
 15. A kit comprising: a) an oligonucleotide labeled with afluorescent dye; and b) an internal standard dye compound; wherein thefluorescent dye of the oligonucleotide and the internal standard dyecompound are spectrally resolvable and further wherein the internalstandard dye compound is:

wherein R₁-R₃ and R₆-R₈ are each independently selected from —H,halogen, aryl, substituted aryl, heteroaryl, —CO₂H, —CO₂R, —SO₃H, —SO₃R,—CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R, —CH₂NH₂, —CH₂NHR, —NO₂, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy,C₁-C₆ alkoxyaryl, substituted C₁-C₆ alkoxyaryl, biphenyl, substitutedbiphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl,wherein R is selected from C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆alkoxy, substituted C₁-C₆ alkoxy, C₁-C₆ alkoxyaryl, substituted C₁-C₆alkoxyaryl, phenyl, substituted phenyl, biphenyl, substituted biphenyl,benzyl, substituted benzyl, benzoyl, and substituted benzoyl; and R₄ andR₅ taken separately are selected from C₁-C₆ alkyl and C₁-C₆ substitutedalkyl, R₄ and R₅ taken together are selected from C₃-C₇ cycloalkyl,C₄-C₇ unsaturated cycloalkyl, C₃-C₇ substituted cycloalkyl and C₄-C₇substituted unsaturated cycloalkyl; with the proviso that at least oneof R₁, R₂, R₃ or R₈ is not —H.
 16. The kit of claim 15, wherein R₆ andR₇ are each independently halogens selected from fluorine, chlorine, andbromine.
 17. The kit of claim 16, wherein R₆ and R₇ are fluorine or R₆and R₇ are chlorine.
 18. The kit of claim 15, wherein R₂ is selectedfrom —SO₃H, —SO₃R, —CH₂NH₂, —CH₂NHR, and —NO₂.